1. Field of the Invention
The invention relates to compositions and methods for generating and using pIX phage display libraries for producing dimeric antibody fragments, whole antibodies, or other disulfide linked multimeric constructs.
2. Discussion of the Field
Filamentous phage display is a widely used technology for affinity-based selection of proteins as each phage particle links the nucleic acid encoding the polypeptide fused to the N-terminus of its coat protein together in the selection process. M13 bacteriophage encodes five coat proteins with approximately five copies of the minor coat proteins pIII and pVI at one end of the phage and the same number of pVII and pIX at other end of the phage. The phage DNA is encapsulated by approximately 3000 copies of the major coat protein, pVIII. Although the display of foreign polypeptides has been accomplished with each of the coat proteins of M13, pIII and pVIII are by far the most common fusion partners. Using this technique, libraries of peptides, Fabs, scFvs and other protein binders have been constructed and found use in diverse applications and with great commercial value.
The pIII coat protein has been favored over the pVIII protein due to its size, conformation and low copy number. The pIII minor coat protein is a 404 amino acid, 42 kD protein responsible for phage infection into E. coli comprising three domains connected by flexible hinge segments. Fusions to the pIII N-terminus tether the displayed protein away from the phage surface, providing potentially greater access for ligand binding than for fusion to the small, high copy number pVIII coat protein. The pIII protein is essential for the initial steps of infection and fusions of all but small peptides and proteins can interfere with this process. This issue is circumvented for example by the use of virus vectors containing a second copy of a wild-type pIII protein or phagemid systems that employ helper phage. In contrast to pIII and like pVIII, pVII and pIX are short helical proteins of 33 and 32 aa, respectively, closely packed on the phage surface. Nevertheless, scFv (Gao, C. et al. Proc Natl Acad Sci USA 99, 12612-12616, 2002) and Fab (Shi, L et al. J Mol Biol 397, 385-396, 2010; Tornetta, M et al. J Immunol Meth 360, 39-46, 2010) libraries have been displayed and selected on pIX. Heterodimeric display of Fv and peptides has been described by fusing different polypeptides to both pVII and the closely adjacent pIX (Gao, et al. 1999 Proc Nat Acad Sci 96: 6025-6030 and Janda U.S. Pat. No. 7,078,166). In addition, pVII display of monospecific scFv has been reported (Kwasnikowski, et al. 2005. J Immunol Methods 307:135). An alternative approach in which exoproteins encoded by the phage or phagemid vector are not fused to the coat protein but rather covalently attach to re-engineered coat proteins pIII and pIX with through disulfide bonding has also been described (U.S. Pat. No. 6,753,136).
The ability to display a dimeric protein on the surface of a phage particle as well as a heterodimeric protein is advantageous in mimicking more complex protein structures in a combinatorial library format. There is a continuing need to advance the art for generating high throughput methods of screening variants of complex proteins such as that of the human IgG, which is a homodimer of heavy and light chain pairs (heterodimers) connected via intermolecular disulfide bonds. To date, it has not been possible to demonstrate the correct assembly and display of complete antibody heavy chains on filamentous phage. The libraries and methods of this invention meet these needs by coupling comprehensive design, assembly technologies, and phage pIX Fab display.